Worldwide, tuberculosis (TB) is the chief opportunistic infection in HIV-infected hosts, as well as the leading cause of death from infectious disease in the population as a whole. The macrophage is the cell responsible for either allowing Mycobacterium tuberculosis (Mtb) to proliferate, or for forcing it into nonreplicative persistence. In the first award period we generated single and double knockout mice whose phenotypes proved the indispensability of two antimicrobial mechanisms of macrophages: production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). We and collaborators showed that Mtb proliferates at its fastest known rate in mice deficient in inducible nitric oxide synthase (iNOS), the source of immunologically induced RNI; that chemotherapy fails to cure mice that lack iNOS; and that iNOS is present in human macrophages from lungs of TB patients. Others confirmed iNOS expression in TB patients' lung macrophages, showed that these cells make large amounts of RNI, and found that human alveolar macrophages can use iNOS to kill mycobacteria. The fact that Mtb nonetheless proliferates to cause TB suggests that disease-causing Mtb may express RNI resistance mechanisms, analogous to known ROI resistance mechanisms like SOD and catalase. In fact, in the first award period we cloned four genes, each of which confers both RNI- and ROI-resistance on bacterial hosts or whose knock-out makes them sensitive to RNI and ROI. Two of these are novel genes found only in Mtb: nitrogen oxides and oxygen intermediates resistance (NOXR)-1 and -3. The other two are widespread, but their role in resistance to RNI was unknown: alkyl hydroperoxide reductase subunit C (AhpC) and peptidyl methionyl sulfoxide reductase (msrA). The goal of this application is to learn how AhpC, NOXR3 and msrA inactivate RNI and ROI, and what overall contribution these gene products make to the ability of Mtb to overcome the host's immune defenses. We will focus first on AhpC, recently shown to be a virulence factor for Mycobacterium bovis, and analyze the impact of mutations, its biochemical mechanism and its expression in Mtb. We will knock out AhpC in Mtb and use wild type, AhpC-deficient and reconstituted Mtb to infect wild type mice and mice genetically deficient in production of RNI, ROI or both. Similar analyses will be done on NOXR3 and msrA. These studies will answer if inhibition of one or more of these gene products might sensitize Mtb to the diminished nitrosative and oxidative attack mounted by the HIV- infected host, and thereby improve the success of residual immunity and chemotherapy.